Selective gene amplification is a widespread phenomenon. Many amplified genes, including oncogenes in tumor cells and many genes of lower eukaryotes, are organized as part of large inverted duplications, or palindromes. Such palindromic sequences are generated from non- palindromic precursors as an initial, and presumably critical, step in gene amplification. Very little is known about how large palindromic DNA is formed. Previous experiments have established that the yeast, Saccharomyces cerevisiae, is an excellent model system for studying palindrome formation. In yeast a linear DNA strand break is introduced next to a 42 base pair (bp) inverted repeat sequence. Conversion of the broken plasmid to a palindrome is strictly dependent on the 42 bp inverted repeats. A mutation in RAD52, a gene required for repair of DNA double-strand breaks (DSBs) by homologous recombination, blocks palindrome formation. The objective of this proposal is to continue the molecular and genetic analysis of DNA palindrome formation in Saccharomyces cerevisiae. The first experiments will address whether palindromic DNA forms by an inter-molecular recombination reaction (that is, a reaction between two identical fragments in a head-to-head arrangement) or by an unusual intra-molecular recombination reaction, as it apparently does in another well studied examined of palindrome formation (see below). The second set of experiments will explore the connections between DSB repair and palindrome formation. Specifically, the effect on palindrome formation of mutations in a variety of DSB repair genes will be tested. The results of these experiments will yield valuable insights into the molecular mechanism and genetic control of this widespread and fascinating type of genome rearrangement.